Ultrasensitive immunoassays

ABSTRACT

The present invention relates to an immunological test kit and immunoassay using a first immobilized antibody having affinity for a specific antigen. The invention is characterized by a second and third antibody being specific for different determinants of the antigen and modified with cross-linkable oligonucleotides. For detection, the oligonucleotides are amplified, whereby only such oligonucleotides will be amplified which have been cross-linked to each other. In this way unspecific background is avoided and detection is possible down to single molecules.

This application is the national phase under 35 U.S.C. §371 of prior PCTInternational Application No. PCT/SE 96/00779 which has an Internationalfiling date of Jun. 14, 1996 which designated the United States ofAmerica, the entire contents of which are hereby incorporated byreference.

TECHNICAL FIELD

The present invention relates to ultrasensitive immunoassays. Morespecifically, it relates to immunological test kits and processes forimmunological detection of a specific antigen. In the present invention,the fields of immunology and molecular genetics are combined.

BACKGROUND OF THE INVENTION

Immunoassays represent powerful tools to identify a very wide range ofcompounds, such as antigens and antibodies. Examples of immunoassays areELISA (enzyme linked immunosorbent assay), EIA (enzyme immunoassay), andRIA (radio immunoassay). Common to all these immunoassays, is thatdetection sensitivity is limited by the affinity of typical antibodies.

With the prior art immunoassays, detection is not possible below acertain number of molecules, because the background, i.e. unspecificallybound material, interferes with the results. Detection of very lownumbers of antigen is becoming increasingly important, especially fordiagnostic applications. Therefore, further developments in sensitivityas well as specificity of immunological assays are desired.

Cantor et al, Science, Vol. 258, 2 October 1992, have previouslyreported the attachment of oligonucleotides to antibodies in order topermit detection of such antibodies having bound antigen in immunereactions. A streptavidin-protein A chimera that posseses tight andspecific binding affinity for both biotin and immunoglobulin G was usedto attach biotinylated DNA specifically to antigen-monoclonal antibodycomplexes that had been immobilized on microtiter plate wells. Then, asegment of the attached DNA was amplified by PCR (Polymerase ChainReaction). Analysis of the PCR products by agarose gel electrophoresisafter staining with ethidium bromide allowed detection of 580 antigenmolecules (9.6×10⁻22 moles) which is a significant improvement comparedto, for example, conventional ELISA.

However, in Cantor et al., the labeled DNA-antibody complexes areassembled in situ during the assay. This can create variablestoichiometry in the assembly of the components and in the attachment ofthe DNA label. Moreover, extra steps are required for addition ofbiotinylated reagents and binding proteins. Numerous wash steps are alsoneeded to remove excess reagents and to free assay components ofnon-specifically bound reagents.

Hendrickson et al., Nucleic Acids Research, 1995, Vol 23, No.3, reportan advancement of the Cantor et al. assay that reduces complexity. Thisis achieved through labeling antibody with DNA by direct covalentlinkage of the DNA to the antibody. In this approach, the analytespecific antibody and the 5′ amino modified DNA oligonucleotide areindependently activated by means of separate heterobifunctionalcross-linking agents. The activated antibody and DNA label are thencoupled in a single spontaneous reaction.

International patent publication no. WO 91/17442 describes a molecularprobe for use as a signal amplifier in immunoassays for detecting i.a.antigens. The probe comprises an antibody, a double strandedpolynucleotide functioning as a promoter for a DNA dependend RNApolymerase, and a single or double stranded template for the promoter.The transcription product is quantified and correlated to the amount ofpresent antigen in a sample.

However, in all three of the above described immunoassays the attachedDNA is only used as a marker by being amplified to detectable levels.There is no distinction between oligonucleotides attached to antibodieshaving bound antigen and oligonucleotides attached to antibodies nothaving bound antigen, i.e. those being non-specifically trapped.Non-specifically trapped antibodies give rise to an undesired backgroundsignal and limits the minimun number of antigen molecules that can bedetected and it will not be possible to distinguish between falsepositive and true positive results below a certain number of antigenmolecules. Commonly, solid supports such as microtiter plates, are usedfor the reactions. According to prior art, there will always be anexcess of oligonucleotide-labeled antibody that cannot be removed fromthe solid support by adding background-lowering agents and by repeatedwash steps.

SUMMARY OF THE INVENTION

The present invention enables detection of extremely low numbers ofantigenic molecules, even down to a single molecule. The inventionprovides reliable immunoassays in situations where insufficient numbersof antigens are available for conventional assays.

According to a first aspect of the invention, there is provided animmunological test kit comprising a first immobilized reagent havingaffinity for a specific macromolecule, such as a protein. Furthermore,the test kit comprises a second and a third affinity reagent specificfor different determinantes of said macromolecule, and modified withcrosslinkable compounds enabling a) conjugation of said second and thirdaffinity reagent only when both are bound to the said, samemacromolecule, and b) detection by amplification.

According to a preferred embodiment of the invention, the affinityreagents are antibodies and the crosslinkable compounds areoligonucleotide extensions attached to the second and third antibody,respectively. The macromolecule is in this case a specific antigen.

According to a second aspect of the invention there is provided animmunoassay for detection of a specific antigen, comprising thefollowing steps:

-   a) contacting a sample suspected of containing said specific antigen    with a first antibody linked to a solid support, said first antibody    being specific for a first epitope on the antigen,-   b) washing off excess reagents,-   c) incubating with a solution of a second and a third antibody    specific for a second and third epitope of said antigen, and    modified with crosslinkable oligonucleotides enabling conjugation of    said second and third antibody when both are bound to the said, same    antigen,-   d) washing off excess reagents,-   e) amplifying said crosslinked oligonucleotides, and-   f) detecting the amplified products.

Products from the amplification reaction only result when twoantibodies, i.e. the second and the third, have bound to the sameantigen. Thus, amplification is specific for antibodies having bound toantigen. Non-specifically trapped antibodies do not give rise to anysignal.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described more detailed below withreference to the accompanying drawings, in which

FIG. 1 is a schematic view of the principles of the immunoassayaccording to the invention, and

FIG. 2 shows chemical coupling of amino-modified oligonucleotides tomacromolecules.

In FIG. 1 there is shown an immobilized antibody to a specific antigenapplied together with two other antibodies, specific for otherdeterminants on the same antigen. Besides antibodies other specificallyinteracting species with a known affinity, such as lectins, receptors,single chain antibodies, cofactors, oligonucleotides and othernon-proteins, can be used in the invention.

The interacting species are modified with crosslinkable compounds in theform of an interacting pair, preferably short oligonucleotideextentions. Upon the coordinated binding of several so modifiedantibodies, oligonucleotides of neighbouring antibodies are conjugatedto each other. The conjugation may or may not necessitate an enzymaticligation step depending on the orientation of the oligonucleotideextensions.

If the conjugation is between free 3′ and 5′ ends ligation is necessary,such as by T4 RNA ligase or T4 DNA ligase. To facilitate theconjugation, it is convenient to use a stretch of oligonucleotides basepairing to and, thereby, juxtaposing the free ends of theoligonucleotides and permitting their joining through ligation.

If the conjugation is between free 3′ ends these have to be designed tobe mutually complementary to achieve base pairing and initiation of DNAsynthesis extending the 3′ ends of the the molecules.

Thus, only in those cases where the antibodies are brought close enoughthrough binding to the same antigen molecule can the oligonucleotides beligated. Ligated molecules subsequently serve as templates for nucleicacid amplification reactions.

In FIG. 2, there is shown a suitable way to attach the oligonucleotideextension to the antibodies. First, the oligonucleotides are terminallyamino-modified and then attached to primary amines on the antibodies viadisulphide bonds, e.g. according to the technique of Chue and Orgel,Nucleic Acid Research, Vol. 16, No. 9, 1988. Another way is by directcovalent coupling as described by Hendrickson et el., supra.

The antibodies used in the invention can be polyclonal, monoclonal orsingle chain antibodies produced by bacteriophages. In the latter case,it is possible to have antibodies equipped with an oligonucleotidebinding part, rendering the above coupling step between antibody andoligonucleotide unnecessary.

The amplification technique to obtain detectable products is, forexample, PCR (Polymerase Chain Reaction), LCR (Ligase Chain Reaction),SDA (Strand Displacement Amplification) bacteriophage Qβ replication,and 3SR (Self-Sustained Synthetic Reaction), of which the latter threemethods do not require temperature cycling.

The method for detecting amplified products can, for example, be directincorporation of a label, such as radioisotopes, fluorochromes, andenzymes, into the amplified products with the use of label-conjugatedprimers or nucleotides. Preferably, the accumulation of amplifiedproducts is monitored via the fluorescence from intercalating dyes, suchas propidium iodide, etidium bromide and SYBR™ green from MolecularProbes.

The invention is not restricted to detection of any special kind ofmacromolecule, such as an antigen; the only criterion it has to fulfilis that it must be able to simultaneously bind three antibodies/affinityreagents. In the case where the affinity reagents are antibodies, thethree antibodies are specific for different epitopes on the antigen. Bybiosensor analysis, it is possible to assure that the antibodies do notbind to overlapping epitopes on the antigen.

Examples of macromolecules are human myoglobin and human growth hormone.Ultrasensitive assays for growth hormone will have significant value inclinical situations where hormone levels are undetectable by prior artassays.

The invention will now be described below in a non-limiting example.

EXAMPLE

Immunoglobulins were modified in a reaction with SPDP(3-(-pyridyldithio) propionic acid N-hydroxysuccinimide ester, fromPharmacia Biotech) according to the manufacturer's suggestions.Oligonucleotides were thiolated, either through the addition of asuitable phosphoramidite according to Connolly (Connolly BA, Nucl. Acid.Res. 1987 15:3131), or 3′aminomodified oligonucleotides were reactedwith SPDP, followed by reduction of the dithiopyridyl bond, usingdithiothreitol.

SPDP-modified antibodies were incubated with three equivalents ofSH-containing oligonucleotides at 4° C. over night. The reaction mixturewas separated using a Zorbax HPLC gel filtration column. Residual freeantibody were removed from the isolated conjugate by ion exchange MonoQFPLC separation.

The two oligonucleotides used to conjugate the antibodies were oligo 1:5′Tr S C3-ATA GAC TGA GCG TGG ACA TTA ATA TGT ACG TAG GCT TTA TTG AGT 3′(SEQ ID NO:1) and Oligo 2: 5′P ATG TAC GAC CCG TAG ATA TTA TCA TAC TGGCAT GGG CAT GAT GAA CAT C-NHSPDP T3′ (SEQ ID NO:2).

The immune test was performed by first binding lpg of biotinylatedantibody (#1) to individual streptavidin-coated prongs on a manifoldsupport. [Parik et al., Anal. Biochem; (1993) 211: 144-150B]. Afterwashes using PBS (phosfihate buffered saline) with 0.5% Tween 20, theprongs were lowered into solutions of antigen (myoglobin) at variableconcentrations. After further washes, the supports with bound antigenwere incubated in a solution of two oligonucleotide-conjugatedantibodies #2 and #3 at 5 ng each per reaction. The supports werewashed, an oligonucleotide complementary to the free ends of theantibody-conjugated oligonucleotides was added (4 pmol per reaction,5′CTA CGG GTC GTA CAT ACT CAA TTA AGC GTA 3′(SEQ ID NO:3)), and the endsof oligonucleotides on nearby antibodies were joined covalently byligation at 37° C. for 30 min using 1 U of T4 DNA ligase. The supportswere then washed in a standard PCR buffer, and the supports were addedas templates in a PCR mix, including two primers specific for sequenceslocated at either side of the ligation junction (5′TTA ATG GCG AG 3′(SEQ ID NO:4)) and Taq polymerase. After two cycles, the supports wereremoved and the amplification was continued for 26 more cycles.Amplification products were examined by separation in an agarose gel andethidium bromide staining.

1. A test kit comprising a) a first immobilized reagent having affinityto a specific macromolecule, and b) a second and a third affinityreagent specific for different determinants of said macromolecule, andmodified with conjugatable oligonucleotides which conjugate through i)hybridization of an oligonucleotide complementary to the conjugatableoligonucleotides; ii) hybridization of the conjugatable oligonucleotidesto each other; or iii) ligation of the oligonucleotides, wherein asignal is generated by nucleic acid amplification only when said secondand third affinity reagents are closely bound on said macromolecule;wherein said macromolecule is a protein.
 2. The test kit according toclaim 1, wherein the affinity reagents are antibodies.
 3. The test kitaccording to claim 1 or 2, wherein the oligonucleotides arecomplementary to each other.
 4. The test kit according to claim 1,further comprising a ligase.
 5. An immunoassay for detection of aspecific antigen, comprising: a) contacting a sample suspected ofcontaining said specific antigen with a first antibody linked to a solidsupport, said first antibody being specific for a first epitope on theantigen; b) washing off excess sample; c) incubating with a solution ofa second and a third antibody specific for a second and a third epitopeof said antigen, and modified with conjugatable oligonucleotides,wherein said oligonucleotides conjugate to each other when said secondand third antibody are both bound to said antigen through i)hybridization of an oligonucleotide complementary to the conjugatableoligonucleotides; ii) hybridization of the conjugatable oligonucleotidesto each other; or iii) ligation of the oligonucleotides; d) washing offexcess solution; e) amplifying said conjugated oligonucleotides; and f)detecting the amplified products.
 6. An immunoassay according to claim5, wherein the conjugation occurs through hybridization of anoligonucleotide complementary to the conjugatable oligonucleotides. 7.An immunoassay according to claim 5, wherein the conjugation occursthrough hybridization of the conjugatable oligonucleotides to eachother.
 8. An immunoassay according to claim 6, wherein the conjugationoccurs through ligation of the oligonucleotides.